适于检测非洲猪瘟病毒的点亮Spinach-p54 RNA适配体的设计及应用  

Design and application of lightening Spinach-p54 RNA apatmer to detect African swine fever virus

作　　者：韩程程 夏凯[1,3] 龚茹莹[1] 吴栩涵 张蕾 梁新乐[1,2] Chengcheng Han;Kai Xia;Ruying Gong;Xuhan Wu;Lei Zhang;Xinle Liang(School of Food Science and Biotechnology,Zhejiang Gongshang University,Hangzhou 310018,China;Institute of Food Biotechnology,Zhejiang Gongshang University,Hangzhou 310018,China;School of Biological and Chemical Engineering,Zhejiang University of Science and Technology,Hangzhou 310023,China)

机构地区：[1]浙江工商大学食品与生物工程学院,杭州310018 [2]浙江工商大学食品生物工程研究所,杭州310018 [3]浙江科技学院生物与化学工程学院,杭州310023

出　　处：《遗传》2021年第12期1170-1178,共9页Hereditas（Beijing)

摘　　要：非洲猪瘟病毒(African swine fever virus,ASFV)是近年来流行的一种主要致病病毒,对我国的国民经济生活造成了严重影响。本研究以非洲猪瘟病毒的编码保守蛋白p54基因序列片段为检测目标,结合点亮Spinach RNA适配体的开关功能,设计了Spinach-p54的嵌合式RNA适配体。其特异地结合目标序列并产生荧光,实现了在RNA水平上对非洲猪瘟病毒的快速检测。研究结果表明,核酸适配体浓度200 nmol/L,反应温度为37℃,该方法检出限为200 nmol/L,线性范围为200~400 nmol/L。该方法特异性强,操作简单,灵敏度高等特点,可用于市场和养殖场的现场快速检测。African swine fever virus(ASFV)is a major and prevalent pathogenic virus in livestock.Its outbreak in the recent years has seriously affected the national economic life.In this study,a specific chimeric RNA aptamer Spinach-p54 corresponding to the conserved protein p54 of ASFV,was designed and used in assembling a toehold RNA module with a truncated lightening Spinach switch.They were used to detect the p54 gene.The aptamer bound rapidly and paired specifically with the ASFV p54 target sequence and produced fluorescence with the addition of DFHBI substrate.The results showed that the limit of detection was as low as 200 nmol/L p54 fragment,under the conditions of 200 nmol/L Spinach-p54 aptamer and 37℃,suggesting that the current method can provide a simple,specific,sensitive,and rapid on-site detection of ASFV at the RNA level in the markets and farms.

关 键 词：核酸适配体 RNA TURN-ON 非洲猪瘟 

分 类 号：S852.651[农业科学—基础兽医学]